PROJECT SUMMARY/ABSTRACT: Chronic kidney disease (CKD) is a growing national health concern affecting 30 million Americans. CKD can lead to permanent loss of kidney function requiring treatment for survival, yet the only two treatment options available are unable to meet the needs of the millions impacted. Engineering a proximal tubule has gained increasing interest as the proximal tubule is responsible for a significant percentage of renal function, and in vitro created proximal tubules have the potential to supplement kidney function. However, current efforts to engineer renal replacement therapy tissue have produced relatively immature tissue in vitro. The majority of these efforts focus primarily on the renal epithelium and vasculature, ignoring a third cell type: the renal interstitium, which we now know plays essential roles in renal development. We discovered that ablation of the renal stroma lead to loss of nephrogenesis and the failure of the collecting duct to mature. Additional studies have shown that the stroma has roles in ureteric bud branching and patterning the vasculature suggesting multiple essential roles in normal renal development. We find that the stroma is molecularly patterned along a cortico-medullary axis into distinct zones that corresponds to the segmentation of the nephron. Each zone shows activity for serval distinct molecular pathways. Perturbations of these pathways lead to a mispatterning of the stroma and impacts the adjacent parenchyma. Based on pervious work and our unpublished observations, we hypothesize that the multiple distinct stroma sub-types provide cell non-autonomous signals that regulate the differentiation and segmentation of the nephron and that when cultured with the proper population of stroma, we can direct the differentiation of a functional proximal tubule. To address these questions, we will characterize stromal heterogeneity and patterning using single cell RNA sequencing and determine the non-cell autonomous impacts of stroma on proximal tubule differentiation and function in vitro using organoid and microfluidic assays and in vivo using CRISPR/Cas9 genetic modification in mouse. These findings will not only provide insight into the functional significance of a patterned stroma on kidney development, they will also aid in the efforts to create functional renal tissue in vitro. This data may help identify unique molecules required for proper nephron differentiation, potentially revolutionizing methods for engineering renal tissue ex vivo. While we acknowledge the possibility that the proximal tubule stroma may have no role in kidney development, it is important to note that it appears that the stromal cells adjacent to the proximal tubules produce EPO. Since CKD patients frequently suffer from anemia, a greater understanding of this cell type is warranted as they may help us to treat CKD and/or anemic patients without recombinant EPO.